1. Field of the Invention
The present invention generally relates to methods of treating hypertension using inhibitors of epoxide hydrolases. Preferred inhibitors include compounds, such as ureas, amides, and carbamates that can interact with the enzyme catalytic site and mimic transient intermediates. Other useful inhibitors include glycodiols and chalcone oxides which can interact with the enzyme as irreversible inhibitors. The invention also relates to methods of identifying patients with disorders or at risk for disorders with abnormal regulation of the cytochrome P450 metabolism or other oxidation of polyunsaturated fatty acids by assaying levels of dihydroxy unsaturated fatty acids.
2. Background of the Invention
The cytochrome P450 monooxygenase pathway metabolizes unsaturated fatty acids. First, epoxy lipids are generated by cytochrome P450 monooxygenase or direct oxidative and peroxidative mechanisms. These lipids are then hydrolyzed by epoxide hydrolase (EH) enzymatically to form dihydroxy unsaturated fatty acids (Zeldin et al. J Biol. Chem. 268:6402-64-07 (1993). These dihydroxy unsaturated fatty acids are in turn substrates for uridine diphosphoglucuronosyl transferases (UDPGTs) and other conjugating enzymes or they can enter beta-oxidation or be incorporated into glycerides. See, FIG. 6. Metabolites from this pathway are known to have potent physiological effects and are associated with diseases such as acute respiratory distress syndrome (ARDS), cardiovascular disease, and hypertension.
The leukotoxin pathway appears to be involved in regulation of vascular permeability, and failure to regulate vascular permeability is associated with a variety of vascular diseases including adult respiratory distress syndrome (ARDS). ARDS is a pulmonary disease that has a mortality rate of 50% and results from lung lesions that are caused by a variety of conditions found in trauma patients and in severe burn victims. (Ingram, R. H. Jr., Harrison""s Principals of Internal Medicine, 13:1240(1995)). In ARDS there is an acute inflammatory reaction with high numbers of neutrophils that migrate into the interstitium and alveoli. If this progresses there is increased inflammation, edema, cell proliferation, and the end result is impaired ability to extract oxygen. The exact cause of ARDS is not known. However it has been hypothesized that over-activation of neutrophils leads to the release of linoleic acid in high levels via phospholipase A2 activity. Linoleic acid in turn is converted to 9,10-epoxy-12-octadecenoate enzymatically by neutrophil cytochrome P-450 epoxygenase and/or a burst of active oxygen. This lipid epoxide, or leukotoxin, is found in high levels in burned skin and in the serum and bronchial lavage of burn patients. Furthermore, when injected into rats, mice, dogs, and other mammals it causes ARDS. With the possible exception of glucocorticoids, there have not been therapeutic agents known to be effective in preventing or ameliorating the tissue injury, such as microvascular damage, associated with acute inflammation that occurs during the early development of ARDS.
Hypertension is the most common risk factor for cardiovascular disease, the leading cause of death in many developed countries. Essential hypertension, the most common form of hypertension, is usually defined as high blood pressure in which secondary causes such as renovascular disease, renal failure, pheochromocytoma, aldosteronism, or other causes are not present (for a discussion of the definition and etiology of essential hypertension see, Carretero and Oparil Circulation 101:329-335 (2000) and Carretero, O. A. and S. Oparil. Circulation 101:446-453 (2000)). Hypertension can also lead to potentially severe complications in human gestation. Pre-eclampsia, eclampsia and pregnancy-induced hypertension (PIH) are characterized by elevated blood pressure, proteinuria and edema. Pregnancy induced hypertension is very common in first pregnancies. Although progression to full eclampsia is rare, morbidity and mortality are very high from this disorder.
A combination of genetic and environmental factors contributes to the development of hypertension and its successful treatment has been limited by a relatively small number of therapeutic targets for blood pressure regulation. Recently, metabolites of unsaturated fatty acids, such as epoxy lipids, have been established as potential targets for hypertensive treatments. For example, renal cytochrome P450 (CYP) eicosanoids, which are metabolites of arachidonic acid, have potent effects on vascular tone and tubular ion and water transport and have been implicated in the control of blood pressure (Makita et al. FASEB J 10: 1456-1463 (1996)). Both major products of CYP-catalyzed arachidonic acid metabolism, regio- and stereoisomeric epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE) have effects on the vascular system. 20-HETE produces potent vasoconstriction by inhibition of the opening of a large-conductance, calcium-activated potassium channel leading to arteriole vascular smooth muscle depolarization (Zou et al. Am J. Physiol. 270:R228-237 (1996)). In contrast, the EETs have vasodilatory properties associated with an increased open-state probability of a calcium-activated potassium channel and hyperpolarization of the vascular smooth muscle and are recognized as putative endothelial derived hyperpolarizing factors (Campbell et al Cir. Res. 78:415-423 (1996)). In fact, recent studies have indicated that renal CYP-mediated 20-HETE and EET formation are altered in genetic rat models of hypertension and that modulation of these enzyme activities is associated with corresponding changes in blood pressure (Omata et al. Am J Physiol 262:F8-16 (1992); Makita et al. J Clin Invest 94:2414-2420 (1994); Kroetz et al. Mol Pharmacol 52:362-372 (1997); Su, P. et al., Am J Physiol 275, R426-438 (1998)).
Modulation of the CYP pathways of arachidonic acid metabolism as a means to regulate eicosanoid levels has been limited by multiple isoforms contributing to a single reaction and the general lack of selectivity of most characterized inhibitors and inducers. Similarly, modulating EET levels by regulation of their hydrolysis to the less active diols has not been considered in light of concerns that EETs are involved in many physiological processes. (Campbell, Trends Pharmacol Sci 21:125-7 (2000)).
In view of the widespread and debilitating effects of these disorders, there is an obvious need for diagnostic tools to identify patients with these diseases or at risk for these diseases. This invention fulfills this and other needs.
The present invention provides methods of treating hypertension by administering to a patient a therapeutically effective amount of an inhibitor of epoxide hydrolase. A preferred class of compounds for practice in accordance with the invention has the structure shown by Formula 1. 
wherein Z is oxygen or sulfur, W is carbon phosphorous or sulfur, X and Y is each independently nitrogen, oxygen, or sulfur, and X can further be carbon, at least one of R1-R4 is hydrogen, R2 is hydrogen when X is nitrogen but is not present when X is sulfur or oxygen, R4 is hydrogen when Y is nitrogen but is not present when Y is sulfur or oxygen, R1 and R3 are each independently a substituted or unsubstituted alkyl, haloalkyl, cycloalkyl, aryl, acyl, or heterocyclic.
Preferred compound of the invention have an IC50 (inhibition potency or, by definition, the concentration of inhibitor which reduces enzyme activity by 50%) of less than about 500 xcexcM. Exemplary compounds of the invention are listed in Table 1. Table shows inhibition of recombinant mouse sEH (MsEH) and Human sEH (HsEH). The enzyme concentrations were 0.13 and 0.26 micromolar respectively
A second preferred class of compounds for practice in accordance with the invention has the structure shown by Formula 2, 
wherein R is alkyl or aryl, the compound is trans-across the epoxide ring, OX is a carbonyl (xe2x95x90O) or hydroxy group (OH) and Rxe2x80x2 is a H, alkyl or aryl group. The preparation of these compounds is described in U.S. Pat. No. 5,955,496 and in WO98/06261.
Exemplary compounds are shown in Table 2, below.
The enzymes of interest for this invention typically are able to distinguish enantiomers. Thus, in choosing an inhibitor for use for an application in accordance with the invention it is preferred to screen different optical isomers of the inhibitor with the selected enzyme by routine assays so as to choose a better optical isomer, if appropriate, for the particular application. The pharmacophores described here can be used to deliver a reactive functionality to the catalytic site. These could include alkylating agents such as halogens or epoxides or Michael acceptors which will react with thiols and amines. These reactive functionalities also can be used to deliver fluorescent or affinity labels to the enzyme active site for enzyme detection.
The present invention provides a method of identifying patients with a disorder or at risk for a disorder with abnormal regulation of cytochrome P450 metabolism or other oxidation of unsaturated fatty acids by measuring dihydroxy unsaturated fatty acid levels. These diseases, include, but are not limited to, acute respiratory distress syndrome (ARDS), cardiovascular disease, and hypertension. In preferred embodiments, the present invention provides a method of identifying a patient at increased risk for these diseases, comprising measuring levels of leukotoxindiol or the respective glucuronide conjugate using an immunoassay. The invention is particularly directed towards identifying patients with an increased risk for pre-eclampsia, eclampsia, and pregnancy-induced hypertension.